Field of the Invention
The present invention resides in the technical fields of molecular biology and diagnostics and relates in particular to amplification of nucleic acids.
Background Art
The detection and/or quantitation of specific nucleic acid sequences is an important technique for identifying and classifying microorganisms, diagnosing infectious diseases, measuring response to various types of treatment, and the like. Such procedures are also useful in detecting and quantitating microorganisms in foodstuffs, water, beverages, industrial and environmental samples, seed stocks, and other types of material where the presence of specific microorganisms may need to be monitored.
Numerous amplification-based methods for the detection and quantitation of target nucleic acids are well-known and established in the art. The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of the target sequence. (See, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 to Mullis et al.; U.S. Pat. No. 5,804,375 to Gelfand et al.; Mullis et al., Meth. Enzymol. 155:335-350, 1987; and Murakawa et al., DNA 7:287-295, 1988).
In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. (See, e.g., U.S. Pat. Nos. 5,322,770 and 5,310,652 to Gelfand et al.)
Another well-known amplification method is strand displacement amplification, commonly referred to as SDA, which uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTP to produce a duplex hemiphosphorothioated primer extension product, endonuclease-mediated nicking of a hemimodified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3′-end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product. (See, e.g., Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U.S. Pat. Nos. 5,270,184 and 5,455,166 to Walker et al.; Walker et al., Nucleic Acids Research 20, 1691-1696, 1992). Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method. (See, e.g., European Pat. No. 0 684 315.)
Other amplification methods include rolling circle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033 to Lizardi); helicase dependent amplification (HDA) (see, e.g., Kong et al., U.S. Pat. Appln. Pub. No. US 2004-0058378 A1); and loop-mediated isothermal amplification (LAMP) (see, e.g., U.S. Pat. No. 6,410,278 to Notomi et al.).
Transcription-based amplification methods commonly used in the art include nucleic acid sequence based amplification, also referred to as NASBA (see, e.g., U.S. Pat. No. 5,130,238 to Malek et al.); methods which rely on the use of an RNA replicase to amplify the probe molecule itself, commonly referred to as Qβ replicase (see, e.g., Lizardi et al., BioTechnol. 6:1197-1202, 1988); transcription-based amplification methods (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-1177, 1989) and self-sustained sequence replication (see, e.g., Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990; Landgren, Trends in Genetics 9:199-202, 1993; and HELEN H. LEE et al., Nucleic Acid Amplification Technologies (1997)).
Another transcription-based amplification method is transcription-mediated amplification, commonly referred to as TMA, which synthesizes multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH, in which multiple RNA copies of the target sequence autocatalytically generate additional copies (see, e.g., U.S. Pat. Nos. 5,480,784 and 5,399,491 to Kacian et al.). TMA is a robust and highly sensitive amplification system with demonstrated efficacy, which overcomes many of the problems associated with PCR-based amplification systems. In particular, temperature cycling is not required.